Hypertension occurs in nearly one out of every three US adults and is a major risk factor for stroke, heart attack, and heart failure. The molecular pathogenesis of hypertension is not well understood;however, recent findings from genetic disorders affecting blood pressure highlight the important roles of the thiazide- sensitive Na-CI cotransporter NCC and the WNK protein kinases in the distal tubule of the kidney in blood pressure regulation. Intriguingly, Na and Ca transport pathways in the distal tubule are inversely related;therefore, an increase in Ca transport in the distal tubule decreases the Na transport and in turn lowers the blood pressure. Hence, a detailed understanding of the interplay between the Na and Ca transport pathways is important in blood pressure regulation. As a gatekeeper for Ca reabsorption, TRPV5 is prominently localized in the apical membrane of the late segment of the distal convoluted tubule, where NCC and WNK4 are also expressed. WNK4 enhances TRPVS-mediated Ca transport in striking contrast to its inhibitory effect on NCC. Furthermore, the enhancing effect of WNK4 on TRPV5 is dose-dependently blocked by NCC. In addition, the enhancing effect of WNK4 on TRPV5 is abolished by chelating intracellular Ca. Based on these findings we hypothesize that WNK4 integrally regulates Na and Ca transport pathways by enhancing TRPV5 and suppressing NCC;the actions of WNK4 are also modulated by TRPV5 and NCC. We plan to test our hypothesis by pursuing three specific aims: 1) determine the mechanism by which WNK4 enhances TRPVS-mediated Ca transport;2) determine the mechanism by which NCC modulates the action of WNK4;and 3) determine the role of Ca/calmodulin in modulating WNK4-mediated regulation. The studies will be carried out using molecular, biochemical and physiological approaches with both X. laevis oocytes and MDCK strain I cells. Stable MDCK I cell lines will be developed to assess the physiological significance of the regulation mechanisms. It is expected that new insights into the regulation of Ca and Na transport pathways through the interplay between the transport proteins and their common regulator WNK4 will be obtained at the completion of the project. With new knowledge acquired from the proposed studies, it is likely a new strategy via integral regulation of Na and Ca pathways in the distal tubule could be developed for blood pressure control.